ABSTRACT Cancer is an exceedingly complex and dynamic disease, and as our knowledge of tumor biology has grown, so has the realization that ever more molecular information is needed to characterize the diverse array of functional states and cell types within heterogeneous tumors. Extracting this information would aid our basic understanding of cancer biology and enable molecular diagnostics that could reveal the underlying driver mechanisms that could be targeted for patient care. This need has driven the current trend towards massive scale ?omics? techniques, such as next generation sequencing and mass spectrometry, but these methods do not offer the cellular resolution or direct functional detail necessary to understand heterogenous systems and identify rare cell types. Imaging platforms based on SERS and mass cytometry have the potential to achieve extremely high numbers of unique probes, but have practical issues in the form of long acquisition times and inherent technological complexity that will limit future clinical adoption. Fluorescence imaging is the most widely used detection technique in biological and clinical settings, and enables fast and simple detection of upwards of ten molecular targets using probes that have different spectral properties. However, a drastic improvement in multiplexing capacity is needed. Fluorescence lifetime is a property that could expand the multiplexing capacity of fluorescence imaging, but to date this approach has been limited to at most two species due to the lack of compatible probes. Here we seek to develop fluorescence lifetime imaging microscopy (FLIM) into a high-content molecular analysis platform from tumor specimens while maintaining the speed and simplicity of traditional spectral fluorescence imaging. To achieve this goal, we will create the first fluorescence ?lifetime probe libraries,? which will emit light in the same spectral window but exhibit unique fluorescence lifetime decays that can be resolved using the powerful phasor approach. We will populate our lifetime libraries by creating a new class of probes that house different components in a modular, flexible nanoparticle format. Specifically, we will encapsulate different fluorescent components at precisely controlled ratios within a silica nanoparticle or shell, which will allow us to tune probe lifetime without affecting emission spectra. This silica-based approach will normalize synthesis and bioconjugation procedures, maximize signal intensity through high loading capacity, be biocompatible, and shield cells from potentially toxic fluorescent components. Critically, the silica shell will also protect the fluorescent components from environmental effects, locking in signal properties. We will first use a panel of four different fluorescent species with similar yellow emission spectra but unique intrinsic lifetimes, and establish methodologies for quantitatively resolving molecular expression levels of cancer cell lines. Next we will create our tunable nanoprobes and construct a library with optimally compatible lifetimes, which we expect will include at least 7 nanoprobes. Finally, we will extend our tunable nanoprobe framework to 4 additional spectral windows, resulting in a combined lifetime and spectral imaging platform with 35 detection channels, and perform a pilot study using human prostate tumor specimens. Our fluorescence lifetime-based molecular imaging platform will be both highly multiplexed while also maintaining the speed and simplicity of traditional fluorescence imaging, which should help drive translation into the clinical arena. Our platform will also be compatible with live or fixed specimens, diagnostic tissue sections, and even in vivo imaging applications. This combination of power, speed, simplicity, and flexibility is not currently available in other high-content molecular analysis platforms.